Castro-Piero et al.

BMC Public Health 2014, 14:400

http://www.biomedcentral.com/1471-2458/14/400

STUDY PROTOCOL Open Access

Follow-up in healthy schoolchildren and in

adolescents with DOWN syndrome:
psycho-environmental and genetic determinants
of physical activity and its impact on fitness,
cardiovascular diseases, inflammatory biomarkers
and mental health; the UP&DOWN Study
Jos Castro-Piero1*, Ana Carbonell-Baeza1, David Martinez-Gomez2, Sonia Gmez-Martnez3,
Vernica Cabanas-Snchez2, Catalina Santiago4, Ana M Veses3, Fernando Bandrs5, Ana Gonzalez-Galo1,
Flix Gomez-Gallego4, Oscar L Veiga2, Jonatan R Ruiz6, Ascensin Marcos3 and the UP&DOWN Study Group

Abstract
Background: An objective diagnosis of sedentary behaviour as well as of the physical activity and fitness levels in
youth and to better understand how lifestyle is associated with cardiovascular disease risk factors and other
phenotypes is of clinical and public health interest, and might be informative for developing intervention studies
focused on the promotion of physical activity in these population. The aim of this methodological paper is to
describe the design and assessment in the UP&DOWN study.
Methods/Design: The UP&DOWN study is a multi-center follow-up design where 2225 Spanish primary and
secondary schoolchildren from Cadiz and Madrid, respectively, as well as 110 Spanish adolescents with Down
syndrome from Madrid and Toledo were recruited to be assessed. Nine main measurement categories are assessed:
i) socio-demographic and early determinants; ii) environmental determinants; iii) physical activity and sedentary
behaviour; iv) health-related fitness; v) blood pressure and resting heart rate; vi) mental health; vii) dietary patterns;
viii) blood samples; and ix) genetic analysis. During the 3-yr follow-up study, socio-demographic and early
determinants, and genetic analysis are only assessed in the first year. Blood sampling is assessed in the first year
and the third year (2nd follow-up), and all the other measurements are assessed every year.
Discussion: The findings of the UP&DOWN study may help the Health Information Systems and policy makers to
identify the target population for primary prevention and health promotion policies, and to develop and test
preventive strategies. Moreover, these data will allow following the trends at population level, as well as to modify/
adapt/create new evidence-based physical activity guidelines at national level. The findings will also serve as a
scientific platform for interventional studies.

* Correspondence: jose.castro@uca.es
1
Department of Physical Education, School of Education, University of Cdiz,
Puerto Real, Spain
Full list of author information is available at the end of the article

2014 Castro-Piero et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the
Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public
Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this
article, unless otherwise stated.
Castro-Piero et al. BMC Public Health 2014, 14:400
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Background
Cardiovascular disease is the leading cause of global mortal-
ity [1]. Cardiovascular disease events occur most frequently
during or after the fifth decade of life, however, there is
evidence indicating that the precursors of cardiovascular
disease have their origin in childhood and adolescence
[2,3]. Adverse cardiovascular disease risk factors during
childhood have been shown to track into adulthood [4].
Therefore, a greater in-depth knowledge of the factors
affecting cardiovascular disease risk factors in youth will
contribute to the development of effective prevention
programs, counselling and public health policy. The pro-
tective effect of physical activity (PA) as well as fitness on
cardiovascular disease risk and mortality has been reported
in people of all ages [5,6].
The determinants of early risk factors for cardiovascular
disease, how they change over time, and how changes in
lifestyle factors affect the risk of cardiovascular disease in
childhood and adolescence is not well understood. There
are reasons to believe that the genotype-environment in-
teractions could also be involved in the susceptibility of
individuals to develop early risk factors for cardiovascular
disease such as insulin resistance, hypertension, dyslipid-
emia, obesity, and chronic inflammation.
Definition of these interaction effects for phenotypes
related to these diseases is therefore important because
it will eventually allow the identification of individuals at
risk of the development of complications and the identi-
fication of those likely to be resistant to lifestyle inter-
ventions. The study of these genetic markers in children
and adolescents and their relationship to several pheno-
typic characteristics of the population will permit a bet-
ter understanding of the pathogenic mechanisms that
are involved in non-communicable diseases, specifically,
cardiovascular disease and diabetes.
Down syndrome (DS) is associated with a trisomy in
chromosome 21. The prevalence of DS is one out of
7001000 live births [7,8]. Fortunately, life expectancy
of DS has increased from 9 years of age in 1929 [9]
to 55 years of age nowadays [7,8]. DS suffer from many
concurrent ailments and there is increasing evidence that
the most common pathologies such as muscle hypotonicity,
hypermobility of the joints or ligamentour laxity, obesity,
undeveloped cardiovascular and respiratory system and
short stature (short legs and arms in relation to torso)
might be modified by increasing levels of PA and fitness
[10]. Assessments of PA in adolescent with DS might be a
challenge using subjective measures and only several at-
tempts have been conducted with objective measures [11].
Similarly, fitness assessments is also complex in this popu-
lation [10]. Studies examining the association of PA and fit-
ness with obesity and related risk factors in this population
are scarce, and the evidence mainly comes from to cross-
sectional studies conducted in relatively small samples.
Page 2 of 12
To have an objective diagnosis of sedentary behaviour
as well as of the PA and fitness levels in youth and to better
understand how lifestyle is associated with cardiovascular
disease risk factors and other phenotypes is of clinical
and public health interest, and might be informative for
developing intervention studies focused on the promotion
of PA in these population.
In the context of a remarkable increase of social
awareness about the problem of physical inactivity in the
population, in May 2009 the Spanish National Sports
Council launched the Integral Plan on Physical Activity and
Sport (A + D Plan). The main aim of this Plan was to drive
coordinated actions to promote PA and sports in the
Spanish population. The plan emphasised the need of
objective information in order to develop nation-wide
promotion strategies. Moreover, in 2005 the NAOS
Strategy (Strategy for Nutrition, Physical Activity and
the Prevention of Obesity) was set up by the Ministry
of Health and Consumer Affairs, through the Spanish
Agency for Food Safety and Nutrition, with the aim of
making the population more aware of the problems
obesity means for health, and of promoting any initia-
tives that help to encourage citizens, particularly chil-
dren and young people, to adopt healthy lifestyles,
mainly through healthy diets and regular physical ac-
tivity. Thus, the UP&DOWN objectives will contribute
to this plan and strategy in several aspects: (i) PA and
health promotion; (ii) promotion of PA and sports in
school population; (iii) promotion of PA and sports in
disabled people; (iv) gender issues related to PA and
sports; and (v) PA and sports in people at deprived
social risk. Therefore, the results obtained from the
UP&DOWN study will be valuable for future develop-
ment of the Spanish A + D Plan, and in addition they
will be helpful to produce relevant scientific informa-
tion and contribute to the NAOS Strategy to inform
public opinion about the risks of obesity and physical
inactivity for health. The purpose of this paper is to
provide an overview on the design, procedures and
methods used in the UP&DOWN study.
Objectives
1. To determine the patterns, interrelationships and
impact on health indicators of objectively and
subjectively measured PA, sedentary behaviours and
health-related fitness (including fatness) in healthy
primary and secondary schoolchildren, and in
adolescents with DS along a 3-yr follow-up.
2. To identify the main psychosocial and
environmental determinants of PA and sedentary
behaviours in healthy primary and secondary
schoolchildren, and in adolescents with DS along a
3-yr follow-up.
Castro-Piero et al. BMC Public Health 2014, 14:400
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3. To investigate genotype-lifestyle interactions in
healthy primary and secondary schoolchildren, and
in adolescents with DS along a 3-yr follow-up.
4. To evaluate the association of PA, sedentary
behaviours and health-related fitness with traditional
and new cardiovascular disease risk factors in
healthy primary and secondary schoolchildren, and
in adolescents with DS along a 3-yr follow-up.
Methods/Design
Study design
The UP&DOWN study is a multi-center longitudinal de-
sign endorsed by the National Research & Development
& Innovation Plan of the Spanish Ministry of Education
and Science, under the Strategic Line of Sport and
Physical Activity.
The management of the UP&DOWN study was designed
to ensure effective collaboration and communication
among the 4 research centers (Spanish National Research
Council (CSIC), University of Cadiz (UCA), Autonomous
University of Madrid (UAM), and Complutense University
of Madrid (UCM)) involved in the longitudinal study. All
centers adhered to a common study protocol for training,
implementation of fieldwork, data collection and manage-
ment, and quality control procedures. At least, once a year
a consortium meeting is organized to analyze the process
and to implement actions resulting from these meetings,
where appropriate.
The UP&DOWN study overall coordination is developed
by CSIC. The principal investigator of each center manages
the day-by-day coordination of their work packages, and
assists the coordinator group on decisions that need to be
taken within a time frame that does not allow a full meeting
of all partners. An UP&DOWN Core Group, composed of
5 members, outlines the scientific strategy, monitors the
progress in view of the overall project timeline and key
milestones, and assists the principal investigators on an ad
hoc basis for all decisions that need to be taken within a
time frame that does not allow a full meeting of all part-
ners, or in cases when immediate contact with one or more
partners is not possible.
Participants and selection criteria
UP&DOWN participants include apparently healthy
Spanish children and adolescents from primary and
secondary schools from regions of Cadiz and Madrid,
respectively, as well as adolescents with DS centers from
regions of Madrid and Toledo. The UP&DOWN study
establishes following inclusion criteria:
Participants selection criteria for healthy children
and adolescents: i) to study in 1st/4th grades
(67 and 910 years old, respectively) for children,
and 7th/10th grades (1213 and 1516 years old,
Page 3 of 12
respectively) for adolescents at baseline; ii) do not
have physical disability or health problems, which
might limit levels of PA.
Participants selection criteria for adolescents with
DS: i) to be 1120 years old with DS; ii) To have an
intelligence quotient over 35; iii) do not have
physical disability for doing PA.
Figure 1 illustrates the participant flow during the re-
cruitment process. An invitation letter to participate in
this study was sent to the headmasters or physical edu-
cation teachers in each school. A total of 23 primary
school, 22 secondary schools and 18 special education
centers, accepted the invitation. Four secondary schools
and two special education centers were excluded for logis-
tical reasons (be located at a great distance from the city or
to have a lower number of students meeting the inclusion
criteria). A meeting with the headmasters was performed to
obtain the centers agreement. Next, all children from 1st
and 4th grades, adolescents from 7th and 10th grades and
adolescents with DS were invited to participate in the study.
The parents of students received a brief flyer describing the
study, including inclusion criteria and an invitation to at-
tend an informative meeting at the school. A meeting
explaining the study purpose was performed in all the cen-
ters and an informed consent for their parents/tutors was
collected. Finally, 1188 children, 1037 adolescents and 110
adolescents with DS agreed to participate in the study.
Outcome measures
During the UP&DOWN study 9 main measurement
categories are assessed: 1) socio-demographic and early
determinants; 2) environmental determinants; 3) PA and
sedentary behaviour; 4) health-related fitness; 5) blood
pressure and resting heart rate; 6) mental health; 7) dietary
patterns; 8) blood samples; and 9) genetic analysis.
All these measurements are assessed in primary school-
children from Cadiz (UCA), and secondary schoolchildren
and DS adolescents from Madrid and Toledo (UAM)
(Figure 2). Blood samples are analyzed in one lab placed
at CSIC (Institute of Food Science, and Technology and
Nutrition), except for haemogram that is analysed by
the same methodologies in two external labs (Cadiz and
Madrid) due to the short spanlife blood cells (Figure 2).
The same methodologies are used in both labs. Saliva sam-
ples are analyzed in Biomedicine laboratory at European
University of Madrid in collaboration with the UCM
(Figure 2). It should be highlighted that all groups have
already collaborated in other co-joint projects (i.e.
AVENA, HELENA, AFINOS, EVASYON).
During the 3-yr follow-up study, socio-demographic
and early determinants, and genetic analysis are only
assessed in the first year (baseline). Blood sampling is
assessed in the first year and the third year (2nd follow-up),
Castro-Piero et al. BMC Public Health 2014, 14:400 Page 4 of 12
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Primary schools invited Secondary schools invited Institutions/organizations invited

n=24 n= 46 n=30

Schools interested in participating Schools interested in participating Centers interested in participating

n=23 n=22 n=17

Schools excluded Centers excluded

n=4 n=2

Meeting with the Meeting with the Meeting with the

Headmaster and obtained Headmaster and obtained Headmaster and obtained
projects approval, n=23 projects approval, n=18 projects approval, n=15

All the students for 1th and 4th All the students for 7th and 10th All adolescents meeting the
grades were invited grades were invited criteria were invited

Informative meeting with parents Informative meeting with

Informative meeting with parents
adolescents and parents

Informed consent to participate Informed consent to participate Informed consent to participate

n=1188 children n=1037 adolescents n= 110 adolescents with DS

Figure 1 Flow diagram of the study participants.

Coordinator: CSIC

UCA UAM

ADOLESCENTS
CHILDREN ADOLESCENTS
( Down syndrome )

- Socio-demographic and early determinants

- Environmental determinants
- Physical activity and sedentary behaviour
- Health-related fitness
- Mental health
- Dietary patterns
- Blood pressure and resting heart rate
CSIC - Blood sampling

UCM - Genetic analysis

Figure 2 Characteristics on management and data collection in the UP&DOWN study.

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and all the other measurements are assessed every year
(Figure 3).
1) Socio-demographic and early determinants
Parents or guardians complete a family questionnaire,
which is a compendium of several questionnaires. Infor-
mation about socio-demographic factors (e.g. sex, age,
birth date, place of birth, time residing in Spain, family
structure) is assessed by standardized questions used in
previous studies [12,13]. Socioeconomic status is assessed
by the Family Affluence Scale [14].
Additionally, parents complete a questionnaire that
will address their educational level and occupation sta-
tus, weight, height, and age, as well as their habitual PA
and sedentary behaviours by the short version of the
International PA Questionnaire [15]. The family ques-
tionnaire also includes questions on several early life
health determinants (e.g. child birth weight and height,
week of gestation, breastfeeding, smoke and PA habits
during pregnancy, motor development during the first
years of life) [16-18].
2) Environmental determinants
Environmental determinants are studied in order to identify
their possible influence on PA and sedentary behaviours.
In this sense, neighbourhood environmental determinants
of PA are assessed using an adapted version of the European
questionnaire developed in the ALPHA project [19]. Schools
environment is evaluated by two instruments based on the
School Health Environment Survey from SHAPES Project
(http://www.shapes.uwaterloo.ca): (i) an audit performed
by the research group and (ii) a questionnaire completed
by participants. Both instruments include questions about
density and use of sports facilities at the school, extracur-
ricular sports programs and competitions, organized PA
Baseline
2011-2012
Cross-sectional
Socio-demographic and early
determinants
Environmental determinants
Physical activity and sedentary
behaviour
Health-related fitness
Blood pressure and resting
hear resting
Mental health
Dietary patterns
Blood analysis
Genetic analysis
Figure 3 Study assessment schedule.
Page 5 of 12
and facilities for active commuting (e.g. bicycle parking,
promoting active commuting, etc.). Some PA barriers such
as parental permissions [20] and accompanied preferences
for active commuting to school are also assessed.
3) PA and sedentary behaviour
The ActiGraph accelerometer models GT1M, GT3X and
GT3X + (Actigraph TM, LLC, Fort Walton Beach, FL, USA)
are used to obtain detailed and objective information about
daily PA and sedentary behaviour over seven consecutive
days. Previous studies have demonstrated that there is
strong agreement between measures from GT1M, GT3X
and GT3X + activity monitors without needing additional
calibration [21,22], as well as they are technically reliable
instruments [23]. The accelerometer is attached tightly in
the hip, on the back side, with the notch faced upwards,
and participants are instructed to use the accelerometer
during waking hours and remove it during water-based ac-
tivities; according to established procedures [24]. The epoch
length is analyzed to 10 seconds to allow a more detailed
estimate of PA intensity. The screening and data processing
procedures to estimate sedentary time, total PA, and PA at
different intensities are consistent with previous studies in
children and adolescents [25-27].
PA is also assessed with the PA Questionnaire for
Adolescents/Children (PAQ-A/C) [28]. This is a self-
administered, 7-day recall instrument, with 9-items
scored in a five-point scale. The PAQ-A/C provides a
final composite activity score, by taking the mean of
the 9 items. This questionnaire has been shown to be
valid and reliable [28,29]. Extracurricular PA is assessed
by the Finnish PA Index [30]. It consists of 5 items
concerning frequency and intensity of extracurricular
PA and participation in organized sports. Using these
five items an index of PA is calculated.
1st follow up 2st follow up
2012-2013 2013-2014
Longitudinal
X
X X X
X X X
X X X
X X X
X X X
X X X
X X
X
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The time spent in sedentary behaviours is assessed by
the Youth Sedentary Behaviour Questionnaire (YSBQ).
The YSBQ was designed in the UP&DOWN framework
to assess the amount of time spent in 16 behaviours that
were proposed previously by Biddle et al. [31]: (i) watching
TV/videos, (ii) playing computer/video games, (iii) inter-
net surfing, (iv) doing homework/study without computer,
(v) doing homework/study with computer, (vi) reading for
fun, (vii) talking on the telephone, (viii) listening to music,
(ix) sitting and talking with family and friends, (x) sitting
to rest, (xi) hanging out, (xii) playing exercise videogames,
(xiii) doing behavioural hobbies, (xiv) doing cognitive
hobbies, (xv) travelling on motorized transport, and (xvi)
doing sports/exercise. The items are completed separately
for weekdays and weekend days, referring to the last week.
Response options are none, 30 minutes, 1 hour, 1 hour
and a half, 2 hours, 2 hours and a half, 3 hours, 4 hours,
and 5 hours or more. Moreover, we created 6 comparative
scales regarding how long participants spend (i) watching
TV/videos, (ii) playing computer/video games, (iii) internet
surfing, (iv) doing homework/study and (v) doing sports/
exercise, and (vi) sitting/lying in comparison with their
peers with same age and sex. The response categories are:
much less than others, some less than others, as well as
others, some more than others and more than others.
Information on the time that their parents, siblings
and friends spend in PA and sedentary behaviours, as
well as the frequency doing PA and sedentary behaviours
with them is also obtained by an internally developed
self-report. Information on how much sport and elec-
tronic equipment children and adolescents have avail-
able at home is obtained. PA facilities checklists were
designed by reference to some previous instruments
[32-34]. Checklist to assess available electronic equipment
at children and adolescents homes and rooms was devel-
oped by adapting an existing instrument by Rosenberg
et al. [35]. The Pros & Cons PACE scale is also used to as-
sess the pros and cons of reducing sedentary behaviours
[36]. This questionnaire asks about the importance of each
statement when deciding whether or not to engage in a
sedentary behaviour, with responses ranging from not
important (1) to extremely important (5). Additionally, we
developed some questions regarding parents restrictions
of sedentary behaviours (e.g. TV watching, telephone/
mobile, console and computer use) separately for weekdays
and weekend days.
4) Health-related fitness
Health-related fitness is assessed by field-based tests and
self-report using the ALPHA health-related fitness test
battery [37,38] and the International Fitness Scale (IFIS)
[39], respectively.
The extended version of the ALPHA health-related
fitness test battery includes the following tests: (i) the
Page 6 of 12
20 m shuttle run test to assess cardiorespiratory fitness; (ii)
the handgrip strength and (iii) standing broad jump to as-
sess musculoskeletal fitness, (iv) the 4 10 m to assess
motor fitness, and (v) body mass index, (vi) waist circum-
ference, and (vii) skinfold thickness (triceps and sub-
scapular) to assess body composition.
(i) 20 m shuttle run test: The participants are required
to run between two lines 20 m apart, while keeping pace
with audio signals emitted from a pre-recorded CD. The
initial speed is 8.5 km/h, and is increased by 0.5 km/h
per minute (one minute equals one stage). Participants
are instructed to run in a straight line, to pivot on com-
pleting a shuttle, and to pace themselves in accordance
with the audio signals. The test will finish when the par-
ticipant fails to reach the end lines concurrent with the
audio signals on two consecutive occasions, or when the
subject stops because of fatigue. The participants are en-
couraged to keep running as long as possible throughout
the course of the test. The test is performed once and
the last completed stage or half-stage at which the sub-
ject dropped out will be scored. A gymnasium or space
large enough to mark out a 20 m track will be used to
perform the test.
(ii) Handgrip strength test: To perform this test a hand
dynamometer with adjustable grip (TKK 5101 Grip D;
Takey, Tokio, Japan) will be used. This dynamometer
presents a high validity and reliability when compared
with calibrated known weights [40]. The participant
squeezes gradually and continuously for at least two sec-
onds, performing the test with the right and left hands
in turn, and with the elbow in full extension as described
elsewhere [40]. The grip-span of the dynamometer is ad-
justed according to the hand size for determining the max-
imum handgrip strength using the equations specifically
developed for children [41] and adolescents [42]. The test is
performed twice and the maximum score for each hand is
recorded in kilograms. The sum of the scores achieved by
left and right hands is used in the analysis.
(iii) Standing long jump test: From a starting position
immediately behind a line, standing with feet approxi-
mately shoulders width apart, the participant jumps as
far forwards as possible on a non-slip hard surface. The
test is performed twice and the best score is recorded in
centimetres.
(iv) 4 10 m shuttle run test: 4 10 m shuttle run is
an adaptation of the 5 10 m shuttle run test included in
the EUROFIT battery [43] but maintaining the same char-
acteristics. Velocity, agility and coordination are assessed in
this test. Participant run 4 10 m (back and forth) as fast
as possible. The test is performed twice and the best score
is recorded in seconds: hundredths.
(v) Body mass index: Participant is barefoot and with
T-shirt and short trousers. Weight is measured with an
electronic scale (Type SECA 861; range, 0.05 to 130 kg;
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precision, 0.05 kg), and stature is measured in the Frankfort
plane with a telescopic stature-measuring instrument
(Type SECA 225; range, 60 to 200 cm; precision, 1 mm).
Body mass index is calculated as weight/height squared
(kg/m2). Participant is categorized according to the BMI
international cut-off values as underweight, normalweight,
overweight, and obesity [44,45]. Weight and stature are
carried out twice, but not consecutively, and mean of the
two measurements is used in the analyses.
(vi) Waist circumference: Waist circumference is
measured with a non-elastic tape (SECA 200; range, 0
to 150 cm; precision, 1 mm), at the level of the natural
waist, in a horizontal plane, which is the narrowest part of
the torso, as seen from a front view. In some obese partici-
pants, it is difficult to identify the waist circumference,
therefore this measurement is performed in the midpoint
between the superior iliac spine and the costal edge in the
midaxillary line [46]. The measurements are taken at the
end of a normal expiration, without the tape compressing
the skin. The measurements are carried out twice, but not
consecutively, and mean of the two measurements is used
in the analyses.
(vii) Skinfolds: Triceps and subscapular skinfold thickness
are measured on the non-dominant side of the body with a
Holtain caliper (range, 0 to 40 mm; precision, 0.2 mm).
Triceps is raised in a vertical fold halfway between the
acromion process and the superior head of the radius, in
the posterior aspect of the arm, and subscapular about
20 mm below the inferior angle of the scapula and 45 to
the lateral side of the body. It is performed according to
Lohmans anthropometric standardization reference man-
ual [47]. Triceps and subscapular skinfold thickness are
carried out twice, but not consecutively, and mean of the
two measurements is used in the analyses.
Additionally, we assess sitting height and neck cir-
cumference. Both, has been associated with cardiovas-
cular risk factors in youth [48,49]. Sitting height is
assessed with the participant sits on the box with his or
her back and buttocks to the backboard of the stadiometer
(Type SECA 225; range, 60 to 200 cm; precision, 1 mm),
and with his or her head in the Frankfort horizontal plane.
The knees are directed straight ahead with the arms and
hands resting at the sides [50]. The measurements are
carried out twice, but not consecutively, and mean of
the two measurements is used in the analyses. Neck
circumference is assessed with the participants stand-
ing in an erect position, hanging their arms freely and
keeping their head aligned in the Frankfort horizontal
plane. The superior border of a non-elastic tape measure
(SECA 200; range, 0 to 150 cm; precision, 1 mm) is placed
just below the laryngeal prominence and applied perpen-
dicular to the long axis of the neck [50]. The measure-
ments are carried out twice, but not consecutively, and
mean of the two measurements is used in the analyses.
Page 7 of 12
The recommended sequence to administer this battery
would be as follows: (1) pubertal status, (2) weight, height
and sitting height (3) waist and neck circumferences, (4)
skinfold thickness (triceps and subscapular), (5) handgrip
strength, standing long jump and 4 10 m shuttle run
test, and (6) 20 m shuttle run test. The ALPHA health-
related fitness test battery also suggest an evaluation of the
pubertal development of children and adolescents in order
to classify themselves in one of the five stages of pubertal
maturity defined by Tanner and Whitehouse [51].
The IFIS is a simple and short self-administered scale
to assess physical fitness. The IFIS is composed of five
Likert-scale questions about the perceived youth overall
fitness and the main components: cardiorrespiratory fit-
ness, muscular fitness, speed-agility, and flexibility in
comparison with their friends physical fitness (very
poor, poor, average, good and very good) [39].
5) Blood pressure and resting heart rate
Blood pressure is measured by a validated digital automatic
blood pressure monitor (OMRON M6, OMRON HEALTH
CARE Co., Ltd., Kyoto, Japan) according to the International
Protocol of the European Society of Hypertension [52].
Resting heart rate is also recorded using the same blood
pressure monitor.
6) Mental health
Positive and negative affect, subjective health, dispositional
hope, health-related quality of life, eating disorders and
academic performance are assessed.
Affectivity is assessed by the PANASN scale of positive
and negative affects for children and adolescents [53].
The PANASN consists of 20 adjective descriptors of gen-
eral mood/affect broken down into two 10-item subscales
of positive affect and negative affect, respectively. Partici-
pants rate each item on a 5-point Likert scale.
Subjective health is assessed by the classic self rated
health status item that consists of asking respondents to
rate their health as excellent, good, fair, bad or poor.
The Childrens Hope Scale assess levels of hopeful
[54]. This scale is comprised of 6-item that assess path-
ways thinking (the childrens perceived capability to
produce routes to those goals) and agency thinking
(perception that children can initiate and sustain action
toward a desired goal). Participants rate each item on a
6-point scale. Health-related quality of life is evaluated by
the KIDSCREEN-10, which consists of a 10-item scale.
Participants rate each item on a 5-point Likert scale [55].
The risk of eating disorders is assessed by the SCOFF
questionnaire, which has been validated for Spanish youth
[56]. It is a screening instrument designed to be routinely
used at population level to identify individuals at risk
for eating disorders such as anorexia nervosa, bulimia,
and binge eating. The questionnaire consists of five
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eating-related questions asking about intentional vomiting,
loss of control over eating, weight loss, body dissatisfaction
and food intrusive thoughts. Answering positively two
or more items of SCOFF questionnaire has been sug-
gested as the threshold for a suspicion of a probable
eating disorder case [57].
Academic performance is assessed through grades
reported by every school. Four main indicators are
used: (i) Mathematics, (ii) Language, (iii) average of
Mathematics and Language, and (iv) grade point average.
Additionally, three school factors related to academic
performance are also obtained (i.e. school attitude, repeating,
absenteeism) [58].
7) Dietary patterns
Both children and adolescents complete a food preference
questionnaire. Information about dietary habits and eating
behaviour (i.e. eating breakfast, meals frequency per day)
is recorded similarly than in previous studies [13,59].
Adherence to the Mediterranean diet is evaluated using
the KIDMED index [60].
8) Blood sampling
A fasting blood sample is obtained from the cubital vein
in the early morning at the schools attended by children
and adolescents in the subset. At least 14 ml of blood
are drawn from each subject and are aliquoted into 3
tubes: 1 tube containing EDTA (2.5 ml), 1 tube sodium
citrate (1.5 ml) and 1 tube containing dry gel for serum
(10 ml). Once the blood is collected, it is immediately
transported to standard laboratories in each city. The
anticoagulated blood in EDTA (2.5 ml) are analysed to
obtain haemogram data. The remainder of the blood
(dried gel and sodium citrate) is centrifuged; serum and
plasma are removed and then frozen at 80C to be ana-
lysed later. The haematological, biochemical and cardio-
metabolic variables measured in the UP&DOWN studies
are shown in Table 1.
9) Genetic analysis
Genomic Deoxyribonucleic acid is extracted from buccal
cells collected on swabs or FTA cards, according to
standard phenol/chloroform procedures followed by
alcohol precipitation.
Allelic discrimination analysis is performed by pre-
designed Life Technologies TaqMan SNP Genotyping
Assays on demand for the fat mass and obesity associated
(FTO) rs9939609 polymorphism (ID: C_30090620_10) and
for the peroxisome proliferator-activated receptor gamma
coactivator 1-alpha (PGC1-) rs8192678 polymorphism
(ID: C_1643192_20).
Polymerase Chain Reaction (PCR) amplification is
performed using a StepOne Real-Time PCR System
(Life Technologies, Foster City, CA) with a denaturation
Page 8 of 12
stage at 95C for 10 min, 50 cycles of denaturation at 92C
for 15 sec, annealing/extension at 60C for 1 min, and a
final extension stage of 30 sec at 60C.
Allelic discrimination analysis for the angiotensin-
converting enzyme (ACE) I/D polymorphism is per-
formed by PCR followed by electrophoresis on a 1.5%
agarose gel containing ethidium bromide. The primers
used are: 5-CTGGAGAGCCACTCCCATCCTTTCT
and 5-GACGTGGCCATCACATTCGTCAGAT [61].
The fragments amplified are a 190 product for allele D
(allele without insertion) and a 490 bp product for allele I
(allele with insertion). In order to avoid a misclassifi-
cation of ID heterozygotes as DD homozygotes, a sec-
ond PCR reaction is performed in all samples initially
classified as DD with these insertion-specific primer
pair: 5-TGGGACCACAGCGCCCGCCACTAC and
5-TCGCCAGCCCTCCCATGCCCATAA [62]. Only the
allele I produced a 335 bp fragment, identified on a 1.5%
agarose gel stained with ethidium bromide performed in
all of the samples initially classified as DD with these.
Other lifestyles and health indicators
Information on the following lifestyles and health indicators
is also obtained by self-report: sleep duration, alcohol and
tobacco consumption, morning and during the day tired-
ness, and time spent with friends per week [63]; back pain-
ful [64]; go late to sleep because of watching TV, and time
spent at home during week and weekend days.
Standardization and harmonization
Both during the feasibility study and for the follow-up
study, there is a strict standardization of the fieldwork.
Methodological coordination and harmonization work-
shops related to the evaluation of questionnaires, the
measurements of anthropometry, PA, fitness, as well as
biological sample transportation and conservation are held
up in order to carry out the same methods, both in children
and adolescents, as well as in adolescents with DS.
To validate and harmonize the methodology, a general
training workshop was held in Madrid (Spain), from 6th
to 7th September 2011. The scientists in charge of every
research tool attended the workshop. Although a general
framework was established for sampling and data collec-
tion, some adaptation was necessary depending on the
local special conditions. The logistic functioning of the
procedure was also tested with a pilot study, which was
organized in Cadiz (children) and Madrid (adolescents),
to check every step of the procedure, from sampling to
data processing.
Finally, a manual of operations was ready to be followed
by all participating research centers. This manual in-
cludes the whole set of data collection methodology
and a detailed description of all instruments for the core
study data. A protocol for data-entry includes specifications
Castro-Piero et al. BMC Public Health 2014, 14:400
http://www.biomedcentral.com/1471-2458/14/400
Table 1 Haematological, biochemical and cardiometabolic variables
Haematological profile
Red blood cell, white blood cell, neutrophil, lymphocyte, monocyte,
basophil, eosinophil, platelet counts, leukocyte profile, haemoglobin
concentrations, haematocrit and haematic indices.
Biochemical variables
Triglycerides, total cholesterol (c), HDL-c, LDL-c, glucose,
and total proteins
Cardio-metabolic variables and inflammatory
Visfatine
TNF-, IL-6, adiponectin, insulin, leptin
Cortisol
C3 and C4 complement factors, and CRP
Fibrinogen
Galectin-3
HDL-c: high density lipoprotein cholesterol; LDL-c: low density lipoprotein cholesterol; TNF-: tumor necrosis factor alpha; IL-6: inteleukin 6; C3: complement factor
3; C4: complement factor 4; CRP: complement factor reactive protein.
aimed at minimising coding errors. Standardised protocols
for the quality control and first round of data cleaning to be
applied by all participating centers were set-up. Central
database architecture for centralisation of data from the
different participating centres and for the different as-
pects of the study is being designed. A central protocol
for anonymous data cross-linkage and a central analytical
plan was developed.
Ethical aspects
This project followed the ethical standards recognized
by the Declaration of Helsinki (reviewed in Seoul, Republic
of Korea in October 2008) and the EEC Good Clinical
Practice recommendations (document 111/3976/88, July
1990), and current Spanish legislation regulating clinical
and biomedical research in humans, personal data pro-
tection and bioethics (Royal Decree 561/1993 on clinical
trials and 14/2007, 3rd July, for Biomedical research). The
study protocols were approved by the Ethics Committee
of the Hospital Puerta de Hierro (Madrid, Spain), the
Bioethics Committee of the CSIC, and the Ethics
Committe for Research Involving Human Subjects at
UCA. The study was explained to the participants before
starting, and the volunteers, parents or tutors signed an
informed consent.
Discussion
Results from longitudinal studies are needed to elucidate
the influence of PA, sedentary behaviours, and fitness
levels at childhood and adolescence on the likelihood of
having disturbances in the cardiovascular disease risk
factors later in life.
Page 9 of 12
Analytical techniques
Automatic cell counter
Analytical techniques
Colorimetric assay (AU2700 Olympus analyser)
Analytical techniques
Enzyme-linked immunoSobent assay (Human visfatin Elisa kit; Cusabio Biotech)
Immunoassay (xMAP Techonology) using a kit (5 + 1) plex: 171B5006M
Bio-Plex Human IL-6 set; 171B5026M Bio-Plex Human TNF- set; 171D50001
Bio-Plex Human Cytokine Stds; 171-A7003M Bio-plex Pro Human Diabetes
Adiponectin Assay; YB0000002Y Bio-Plex Human Diabetes 3-Plex Assay
Enzyme-Linked ImmunoSorbent Assay (ARBOR ASSAYS kit)
Turbidimetry (AU2700 Olympus analyser)
Clauss method
Enzyme-Linked ImmunoSorbent Assay (Omnikine TM Human Galectin- 3
Elisa Kit, assay biotech)
Studying the genetic markers related to different car-
diovascular disease risk factors and their relationship
to several phenotypic characteristics of the population
will establish a new and better understanding of: 1)
the pathogenic mechanism that are involved in non-
communicable diseases, that is, a better understanding
on the disease aetiology; 2) to refine the genetic and
environmental effects; 3) to identify individuals at risk
of the development of complications and the identifi-
cation of those likely to be resistance to interventions;
4) to inform strategies of targeted disease prevention.
The findings of the UP&DOWN study may help the
Health Information Systems and policy makers to identify
the target population for primary prevention and health
promotion policies, and to develop and test preventive
strategies. Moreover, these data will allow following the
trends at population level, as well as to modify/adapt/
create new evidence-based PA guidelines at national level.
The findings will also serve as a scientific platform for
interventional studies.
Abbreviations
ACE: Angiotensin converting enzyme; AFINOS: Physical Activity as a
Preventive Measure for Overweight Obesity Infection Allergies and
Cardiovascular Risk Factors in Adolescents; ALPHA: Assessing Levels of
Physical Activity and Fitness; DS: Down syndrome; CSIC: Spanish National
Research Council; EDTA: Ethylene diamine tetraacetic acid; EUROFIT: European
Physical Fitness Test Battery; FTO: Fat mass and obesity associated; HDL-c: High
density lipoprotein cholesterol; IFIS: International Fitness Scale; LDL-c: Low density
lipoprotein cholesterol; PA: Physical activity; PANASN: Positive and Negative Affect
Schedule for children and adolescents; PAQ-A/C: Physical Activity Questionnaire
for Adolescents/Children; PCR: Polymerase chain reaction; PGC1-: Peroxisome
proliferator-activated receptor gamma coactivator 1-alpha; SHAPES: School Health
Action Planning and Evaluation System; UAM: Autonomous University of Madrid;
UCA: University of Cadiz; UCM: Complutense University of Madrid; YSBQ: Youth
Sedentary Behaviour Questionnaire.
Castro-Piero et al. BMC Public Health 2014, 14:400
http://www.biomedcentral.com/1471-2458/14/400
Competing interests
The authors declare that they have no competing interests.
Authors contributions
JCP, OLV, JRR and AM conceived the study and obtained funding. JCP, ACB,
DMG and JRR drafted the manuscript. ACB, DMG and JRR contributed
equally to this work. SGM, VCS, CS, AMV, FB, AGG, FGG, OLV and AM
participated in the study design and critically revised the manuscript. Final
approval of the version to be published was obtained from each of the authors.
Acknowledgements
We gratefully acknowledge the participation and collaboration of all primary
and secondary schoolchildren and adolescents with DS as well as all their
families in this study. This study was supported by the DEP 2010-21662-C04-00
(DEP 2010-21662-C04-01, DEP 2010-21662-C04-02, DEP 2010-21662-C04-03, DEP
2010-21662-C04-04) RYC-2010-05957 grants from the National Plan for Research,
Development and Innovation (R + D + i) MICINN.
The UP&DOWN Study Group
Coordinator: Ascension Marcos.
Principal Investigators: Ascension Marcos, Jose Castro-Piero, Oscar L. Veiga
and Fernando Bandres.
Scientific coordinators: David Martinez-Gomez (chair), Jonatan R. Ruiz (co-chair),
Ana Carbonell-Baeza, Sonia Gomez-Martinez, and Catalina Santiago.
Spanish National Research Council: Ascension Marcos, Sonia Gomez-Martinez,
Esther Nova, Ligia-Esperanza Diaz, Belen Zapatera, Ana M. Veses, Jorge R.
Mujico, Aurora Hernandez and Alina Gheorghe.
University of Cadiz: Jose Castro-Piero, Jesus Mora-Vicente, Jose L. Gonzalez-
Montesinos, Julio Conde-Caveda, Jonatan R. Ruiz (University of Granada
(UGR)), Francisco B. Ortega (UGR), Carmen Padilla Moledo, Ana Carbonell
Baeza, Palma Chillon (UGR), Jorge del Rosario Fernandez, Ana Gonzalez Galo,
Gonzalo Bellvis Guerra, Alvaro Delgado Alfonso, Fernando Parrilla, Roque
Gomez, and Juan Gavala.
Autonomous University of Madrid: Oscar L. Veiga, H. Ariel Villagra, Juan
del-Campo, Carlos Cordente (UPM), Mario Diaz, Carlos M. Tejero, Aitor Acha,
Jose M. Moya, Alberto Sanz, David Martinez-Gomez, Veronica Cabanas-Sanchez,
Gabriel Rodriguez-Romo (UPM), Rocio Izquierdo, Laura Garcia-Cervantes, and
Irene Esteban-Cornejo.
Complutense University of Madrid: Fernando Bandres, Alejandro Lucia
(European University of Madrid (UEM)), Catalina Santiago (UEM), and Felix
Gomez-Gallego (UEM).
Author details
1 Pratt M, Ekelund U, Yngve A, Sallis JF, Oja P: International physical activity
Department of Physical Education, School of Education, University of Cdiz,
Puerto Real, Spain. 2Department of Physical Education, Sports and Human
Movement, Faculty of Teacher Training and Education, Autonomous
University of Madrid, Madrid, Spain. 3Immunonutrition Research Group.
Department of Metabolism and Nutrition. Institute of Food Science and
Technology and Nutrition (ICTAN), Spanish National Research Council (CSIC),
Madrid, Spain. 4School of Doctoral Studies & Research, European University,
Madrid, Spain. 5Department of Toxicology and Health Sanitary, Complutense
University of Madrid, Madrid, Spain. 6PROFITH PROmoting FITness and
Health through physical activity research group, Department of Physical
Education and Sport, Faculty of Sport Sciences, University of Granada,
Granada, Spain.
Received: 28 March 2014 Accepted: 14 April 2014
Published: 25 April 2014
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doi:10.1186/1471-2458-14-400
Cite this article as: Castro-Piero et al.: Follow-up in healthy
schoolchildren and in adolescents with DOWN syndrome:
psycho-environmental and genetic determinants of physical activity and
its impact on fitness, cardiovascular diseases, inflammatory biomarkers
and mental health; the UP&DOWN Study. BMC Public Health 2014 14:400.

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